PROJECT SUMMARY/ABSTRACT Orofacial clefts are one of the most common birth defects, affecting about 220,000 newborns each year. The cause and prevention of orofacial clefts remain poorly understood. We have uncovered that the Lrp6-mediated Wnt/-catenin signaling pathway is required for lip and palate formation and fusion, which may act through positive regulation of the Msx homeobox-containing genes, and may repress a retinoic acid-synthesizing enzyme in the orofacial primordia. Orofacial development involves both orofacial mesenchymal expansion and ectodermal/epithelial fusion processes. We demonstrated that either loss- or gain-of-function of facial ectodermal -catenin signaling arrested the formation and patterning of orofacial primordia, which may act through transcriptional regulation of Fgf8, a critical signaling molecule in the facial ectoderm and anterior neural ridge (a regional signaling center). Loss- or gain-of-function of palatal epithelial -catenin caused cleft palate. These studies suggest that Wnt/-catenin signaling activity is tightly regulated during normal orofacial morphogenesis and that either hypo- or hyperactivity of Wnt/-catenin signaling may cause orofacial birth defects. However, the regulatory mechanism of Wnt/-catenin signaling pathway during orofacial development remains almost unknown and will be addressed in this study. Orofacial clefts can be caused by gene mutations and/or environmental factors. The latter may affect epigenetic processes, such as DNA methylation and chromatin modification, to regulate gene expression activities without altering the genomic sequence. Nevertheless, epigenetic mechanisms of orofacial clefts remain poorly understood. We hypothesize that epigenetic factors regulate appropriate Wnt/-catenin signaling activities for lip/palate formation and fusion. Specific Aim 1 will address the role of two epigenetic regulators that may lead to orofacial clefts if they are not properly regulated. Specific Aim 2 will test genetic interactions and/or the genetic rescue of key Wnt/-catenin signaling components and the epigenetic factors in orofacial clefts. Specific Aim 3 will test the hypothesis that epigenetic modulation of Wnt/-catenin signaling can prevent orofacial clefts in mutant animal models. Successful completion of the proposed research will provide new insights into the genetic and epigenetic mechanisms of orofacial clefts, which may translate into clinical applications to treat orofacial birth defects through manipulation of key regulatory processes.